Support for growing/regenerating plant and method of growing/regenerating plant

ABSTRACT

A plant is grown or regenerated while suppressing the propagation of bacteria and fungi, by use of a carrier comprising a culture medium and a polymer constituting a network structure wherein the culture medium is substantially absorbed into and retained by the network structure in a proportion of 10% to 100% of the equilibrium culture medium absorption of the polymer constituting the network structure. The propagation of bacteria and fungi is much faster, and the metabolic rate thereof is much larger, than those of a plant. Therefore, the necessity of providing a nutrient (such as water and saccharide) from the culture medium to the bacteria and fungi is much greater than that in the plant tissue. Unlike a liquid medium, the culture medium which has been absorbed in the network structure comprising the polymer is hardly available to the bacteria and fungi, which have high metabolic activity. On the other hand, like a liquid medium, such a culture medium is available to a plant tissue having a low metabolic activity. The propagation of bacteria and fungi is effectively substantially suppressed without affecting the growth or regeneration of the plant.

TECHNICAL FIELD

The present invention relates to a support (or carrier) which issuitable for the growth or regeneration of a plant. More specifically,the present invention relates to a method of promoting or acceleratingthe growth or regeneration of a plant while inhibiting the propagationof bacteria and fungi, by using a support for growing or regeneratingthe plant which is capable of effectively inhibiting the propagation ofbacteria and fungi; and/or a support for growing or regenerating a plantwhich is capable of easily collecting or transferring (subculturing) theplant; and a method of growing or regenerating a plant by utilizing sucha support.

BACKGROUND OF THE INVENTION

In recent years, it has attracted much attention to develop a techniquefor growing or regenerating a plant having a character suitable for anintended purpose.

Quite recently, on the basis of a technique for growing a plantlet froma growing (or vegetative) point, have been various e.g., those forregenerating a plant from an organ such as leaf, stem, root, petal, andanther (pollen) directly, or by way of a callus or protoplast.Particularly, at the stage of the protoplast, new breeding techniquessuch as the introduction of a foreign or exogeneous gene using aparticle gun and electroporation have been developed, and there havebeen attempts to grown and regenerate a plant which is more suitable foran intended purpose.

In a case where these new breeding or plant tissue-culturing processesare put to practical use, it is the most important point, commonly inthe above techniques, to establish a method of efficiently growing orregenerating a plant which has acquired the above-mentioned intendedcharacter.

In view of the physical property of a "culture medium" to be used forthe culture process, the plant tissue-culturing processes are roughlyclassified into a method (liquid culture process) using a liquid culturemedium, and a method (solid culture process) using a culture mediumwhich has been converted into a gel state by using agar, gellangum(trade name: GELRITE, e.g., mfd. by San-ei Kagaku Kogyo K.K.), etc.

The liquid culture process in which a plant tissue or cells are grown ina suspension state, may facilitate the rapid and large-quantitymultiplication thereof, and therefore such a method is suitable for abioreactor, etc., which is to be used for the purpose of producing asecondary metabolite of a plant. However, in many cases, the liquidculture process is not suitable for the growth or regeneration of aplant which is intended for the multiplication of a clone. The reasonfor this is that, in the liquid culture process, a plant is grown in astate such that the plant is soaked in a culture medium, and thereforethe resultant plant has an extremely poor resistance to dryness, and itis impossible to transfer the resultant plant into field (or farm)cultivation which is to be conducted at a relatively low humidity.

On the other hand, in the solid culture process using agar gel, etc.,the gel state of a culture medium functions as a good support for aplant, and therefore such a method may solve the above-mentioned problemof "soaking in liquid" which will occur in the case of the liquidculture process. Accordingly, the solid culture process has widely beenused for growing or regenerating a plant.

However, with respect to the current process (for growing orregenerating a plant (i.e., solid culture process) by using agar, etc.,some serious problems as described below have been pointed out.

Thus, in the process for growing or regenerating a plant by using agar,it is extremely difficult to collect (or harvest) the resultant plant,which has been grown or regenerated in the gel-like support such as agargel, in a state such that the plant is free from the agar gel.Particularly, when some roots are regenerated in the gel-like agar inthe step of the formation of roots originating from a shoot, it isextremely difficult to separate such roots from the agar. The agarculture medium to be used for the root-forming step usually contains asaccharide or sugar such as glucose, as a nutrient. Therefore, when theresultant plant having the thus formed roots is as such subjected tocultivation outside a culturing vessel, i.e., in a state such that theagar is attached to the roots, under a non-sterilized condition, thesaccharide contained in the agar becomes a cause of the propagation ofbacteria. As a result, the resultant efficiency of active root anchoringis markedly decreased, in a case where the root-originating plant issubjected to the cultivation outside the culturing vessel.

At present, there has been adopted a method wherein the agar ismechanically removed from the root-originating plant, and then is washedout with water for the purpose of removing the agar from the plant.However, in such a procedure wherein the agar is mechanically removed,not only are the weak or fragile roots of the root-originating plant tobe collected damaged, but also it is difficult to completely remove theminute pieces of the agar gel which have firmly been attached to theroots. As a result, the efficiency of the active root anchoring of thecollected plant is inevitably decreased. In addition, the aboveagar-removing step requires much labor. Such a step becomes a factor ofan increase in the production cost.

The above-mentioned problems are based on the physical property of thegel such as agar gel to be used for the conventional solid cultureprocess. More specifically, the agar has a sol-gel transitiontemperature, and has a property such that it assumes a solution state,namely, a sol state, at a temperature higher than the transitiontemperature, and it is converted into a gel state at a temperature lowerthan the transition temperature. Accordingly, the solid culturingprocess has been practiced, e.g., by using a method wherein an agar gelis formed at a temperature lower than the sol-gel transitiontemperature, and then a plant is transferred into the resultant agargel.

However, the melting temperature of the agar gel is very high, i.e., inthe neighborhood of 90° C. (Aizo Yamauchi, et al., KOBUNSHI (Polymer)One Point "Functional Gel", page 29, Kyoritsu Shuppan K.K.). Therefore,when a plant, which has been grown or regenerated in the agar culturemedium, is intended to be collected in an agar-free state, it isnecessary to raise the temperature up to a value higher than the sol-geltransition temperature of agar, so as to convert the agar into a solstate. However, at this time, the plant is exposed to such a hightemperature and is seriously damaged. Accordingly, in the conventionalsolid culture process, it is extremely difficult to collect the plant inan agar-free state by utilizing a temperature change as described above,and therefore the above-mentioned method wherein the agar ismechanically removed has been practiced. However, in the mechanicalagar-removing method, some serious problems still remain unsolved, asdescribed above.

In addition, in the course of the solid culture process, a nutrient issupplied to the agar culture medium, and the resultant waste (or egesta)accumulated in the agar culture medium is removed therefrom, both on thebasis of the diffusion of the nutrient or waste in the gel. Therefore,the efficiency in the supply of the nutrient and in the removal of thewaste is very low, as compared with that in the case of the liquidculture process. Particularly, it is almost impossible in the solidculture process to remove a growth inhibiting substances such aspolyphenol produced by the plant per se.

In addition, the collection or recovery of a secondary metabolite of aplant in the agar culture medium is much harder than that in the liquidculture process. More specifically, the reason for this is that thesecondary metabolite is seriously damaged inevitably by thehigh-temperature heating to be employed at the time of dissolving theagar gel containing the secondary metabolite.

On the other hand, the plant (cell) culture processes for the purpose ofgrowing or regenerating a plant may generally be classified into asaccharide-involving (or saccharide-relating) culture process and asaccharide-free culture process, in view of the supply of a nutrient.

In the saccharide-involving culture process, a saccharide such assucrose, glucose, and fructose is added into the culture medium (orsupport) as a nutrient, and therefore the saccharide-involving cultureprocess is suitable for the culture of a plant at a stage at which theleaf thereof capable of photo-synthesis is still small, i.e., a youngplant. On the other hand, the saccharide-free culture process issuitable for the culture of a plant at a stage at which the leaf thereofcapable of photo-synthesis becomes large, i.e., a grown plant. In thesaccharide-free culture process, in general, the plant is supplied withcarbon dioxide and is irradiated with light so as to promote thephoto-synthesis reaction.

In the above-mentioned saccharide-involving culture process, since thesaccharide contained in the culture medium increasingly promotes thepropagation of bacteria and fungi, the contamination of the culturemedium with various germs will provide fatal results. Accordingly, thesaccharide-involving culture process has heretofore been conducted undera strictly sterilized environment.

On the other hand, in the saccharide-free culture process, thepropagation of bacteria and fungi is relatively slow as compared withthat in the saccharide-involving culture process. However, even in thecase of the saccharide-free culture process, the contamination ofculture medium may possibly provide fatal results similarly as in thecase of the saccharide-involving culture process. From such a viewpoint,in practice, the saccharide-free culture has heretofore been conductedunder a sterilized or closed environment.

In the above-mentioned sterilized culture process wherein thecontamination with bacteria must be minimized, there have been posedserious problems such that a costly apparatus is required in order toprovide the sterilized environment; operations for sterilization ordisinfection of various devices, a culture medium, etc., to be requiredfor the procedure are extremely complicated and troublesome; and most ofthe operations per se for the transferring of a plant, etc., themselvesdepend on human labor and the saving of the labor in these operations isdifficult.

Further, in the solid culture process using an agar gel, along withprogress in the growth or regeneration of a plant, an organ (such asroot) of the resultant plant tissue generally destroys the agar gel soas to provide voids between the agar gel and the plant (in some cases,these voids are filled with a liquid culture medium), when such an organof the plant tissue is grown and penetrated into the agar gelfunctioning as the support for the plant. The cause for this phenomenonis considered to be that the agar gel has a crosslinked networkstructure in the interior thereof, but the growing or regenerating plantcannot penetrate into the network crosslinking structure, and thereforethe plant inevitably destroys the agar gel so that the plant tissue isgrown in the culture medium. Based on the property of the agar gel, thevoids which have been provided between the plant tissue and the agar gelcannot be filled with the agar, unless the agar gel is again convertedinto a sol state. However, the melting temperature of the agar gel is ashigh as about 90° C., and therefore it is practically impossible toconvert the agar gel into a sol state so as to fill the above-mentionedvoids in the agar (in consideration of the prevention of the thermaldamage to the plant tissue).

In addition, the agar gel, once formed, has a characteristic such thatit does not absorb a further amount of the culture medium even if theculture medium is newly added to the agar gel. In combination with suchan additional characteristic of the agar gel, it is impossible to fillthe above-mentioned voids which have been formed between the plant andthe agar gel, and further the culture medium occupying the voids is notabsorbed into the gel. In other words, even in the solid culture processusing the agar gel, the environment surrounding the plant tissue isequivalent to that in the liquid culture process under microscopicobservation. In addition, it has been pointed out that the agar gel hasa serious problem such that it may cause a syneresis phenomenon whereinthe culture medium is separated from the agar gel, and bacteria andfungi are rapidly propagated through the culture medium which has beenseparated from the gel.

Accordingly, even the agar gel culture process has a serious problemsuch that once the culture medium is contaminated, bacteria and fungicannot penetrate into the interior of the agar gel, but the bacteria andfungi are extremely rapidly propagated through the voids which have beenprovided between the plant and the agar gel, or through the culturemedium which has been separated from the agar gel.

An object of the present invention is to provide a support (or carrier)for growing or regenerating a plant which promotes the growth of theplant while effectively suppressing the propagation of bacteria andfungi; and a process for growing or regenerating a plant which enablesthe saving of labor by using such a support.

Another object of the present invention is to provide a support (orcarrier) for growing or regenerating a plant which can easily collect ortransfer the grown or regenerated plant without damaging the plant; anda process for growing or regenerating a plant by using such a support.

A further object of the present invention is to provide a support (orcarrier) for growing or regenerating a plant, from which a saccharidesuch as glucose, which is a nutrient for bacteria and fungi, may beremoved to the outside of the plant-growing/regenerating system, whenthe plant which has been grown or regenerated under a sterilizedenvironment is transferred into a non-sterilized environment such asfield or farm; and a process for growing or regenerating a plant byusing such a support.

DISCLOSURE OF INVENTION

As a result of earnest study, the present inventor has found that it isextremely effective in solving the above-mentioned problems by usinghydrogel particles having a predetermined size in a dried state thereofand also having a crosslinked structure, as a support for growing orregenerating a plant.

The support for growing or regenerating a plant according to the presentinvention is based on the above discovery and comprises: particleshaving a dimension in the range of 0.1 μm to 1 cm in a dried state, andcomprising a hydrogel having a crosslinked structure (Hereinbelow, thesupport in such an embodiment is sometimes referred to as "supportaccording to first embodiment").

The present invention also provides a support for growing orregenerating a plant, wherein a grown or regenerated tissue of the plantdoes not penetrate into the interior of the hydrogel particle having acrosslinked structure.

The present invention further provides a support for growing orregenerating a plant, wherein the hydrogel particles are in the form of:micro-beads, fibers, flakes, a sponge, a film or a plate (indeterminateshape).

The present invention further provides a method of growing orregenerating a plant, wherein a support is swollen with a culture mediumin a culturing vessel so as to reduce the fluidity of the culture mediumto be formed into a gel state, and to support a plant by the gel,thereby to grow or regenerate the plant; the support comprisingparticles having a dimension in the range of 0.1 μm to 1 cm in a driedstate, and comprising a hydrogel having a crosslinked structure.

The present invention further provides a method of growing orregenerating a plant, wherein the plant is grown or regenerated by usingthe support, and thereafter an excess of water is added to the supportto increase the fluidity of the support, thereby to recover the plant.

The above-mentioned support according to the first embodiment is oneutilizing a property of the hydrogel particles having a size in therange of 0.1 μm to 1 cm in a dried state and having a crosslinkedstructure are swollen in water or a culture medium so that their volumeis reversibly increased. Herein, the term "hydrogel" refers to a gelcomprising, at least, a crosslinked water-soluble or hydrophilicpolymer, and water as a dispersion liquid (or a liquid comprising wateras a main component) supported by the polymer.

The above-mentioned support according to the present invention is oneobtained by crosslinking a water-soluble or hydrophilic polymer so thatwhen the resultant polymer is placed in a solution, it absorbs water tobe swollen, but is not dissolved therein. The degree of the swelling ofsuch a support may be changed by changing the kind of the water-solubleor hydrophilic polymer, and/or the degree of the crosslinking thereof,etc. In the present invention, it is preferred to regulate the abovedegree of crosslinking so that the grown or regenerated tissue of aplant cannot penetrate into the inside of the crosslinked networkstructure of the hydrogel particle according to the present invention,but the tissue is propagated along the clearances between the abovehydrogel particles. The ability of the support according to the presentinvention for supporting a plant in the course of the growth orregeneration thereof can be regulated by the above-mentioned degree ofswelling, the volume ratio between the above support and culture mediumin a culture vessel (culture system), the shape and dimension of theabove support, etc.

On the other hand, when the plant which has been grown or regenerated byusing the above support is collected (or harvested) from the support,for example, it is easy to separate the plant from the support by addingan excess of water or a culture medium into the culture vessel so as todilute the support contained in the culture vessel (i.e., to decreasethe volume ratio of the support to the culture medium) and to decreasethe plant-supporting ability of the support.

As a result of further study of the present inventor, it has also beenfound that the above-mentioned problems encountered in the prior art aresolved extremely effectively by using a polymer which is capable ofreversibly converting between a liquid state and a gel state (i.e., atemperature-responsive polymer having a LCST (lower critical solutiontemperature)) as a support for growing and/or regenerating a plant.

The support for growing or regenerating a plant according to the presentinvention is based on the above discovery and comprises: a polymer whichhas been obtained by crosslinking a temperature-responsive polymerhaving a LCST (lower critical solution temperature). (Hereinafter, thesupport according to such an embodiment is sometimes referred to as"support according the second embodiment".) With respect to the detailsof the above "LCST", e.g., a paper of D. Patterson; Macromolecules, 2,1672 (1969) may be referred to.

The present invention further provides a method of growing orregenerating a plant, comprising:

dispersing a carrier for growing or regenerating a plant, which has beenobtained by crosslinking a temperature-responsive polymer having a LCST(lower critical solution temperature), in a predetermined culture mediumat a temperature higher than the LCST;

mixing a plant in the resultant dispersion; and

lowering the temperature to a value lower than the LCST to reduce thefluidity of the dispersion and to convert the dispersion into a gelstate, thereby to grow or regenerate the plant.

The present invention also provides a method of growing or regeneratinga plant, comprising:

dispersing a carrier for growing or regenerating a plant, which has beenobtained by crosslinking a temperature-responsive polymer having a LCST(lower critical solution temperature), in a predetermined culture mediumat a temperature higher than the LCST;

lowering the temperature to a value lower than the LCST to reduce thefluidity of the resultant dispersion and to convert the dispersion intoa gel state; and

disposing or inserting a plant on or in the gel, thereby to grow orregenerate the plant.

The above support according to the second embodiment is one whichutilizes a property such that a polymer obtained by crosslinking atemperature-responsive polymer having a LCST absorbs water at atemperature lower than the LCST in water or a culture medium so as to beswollen, and also releases the water at a temperature higher than theLCST to be shrunk so as to markedly change the volume thereof.

Accordingly, both of a gel state having an extremely low flowability ata temperature lower than the LCST, and a liquid state having anextremely high flowability at a temperature higher than the LCST may berealized, e.g., by controlling the quantitative ratio of the support andthe culture medium in the culture medium dispersion of the carrieraccording to the present invention, the degree of crosslinking of thetemperature-responsive polymer, the dimension of the support, etc. Sucha conversion between the liquid state and the gel state is reversiblewith respect to temperature. Further, the temperature for causing theconversion between the liquid state and the gel state may be determinedby the above LCST.

As a result of further study of the present inventor, it has been foundthat the use of a carrier obtained by causing a culture medium to besubstantially absorbed and retained in the network structure of apolymer (e.g., in the network structure constituting the above-mentioned"first support" and/or "second support") enables the growth orregeneration of a plant while remarkably suppressing the propagation ofbacteria, fungi, etc., thereby to provide a further effect on theachievement of the above-mentioned objects.

The support for growing or regenerating a plant is based on the abovediscovery and comprises: a culture medium and a polymer, wherein theculture medium is substantially absorbed into and retained by thenetwork structure comprising the polymer.

In the above-mentioned carrier, it is preferred that the above networkstructure constitutes a hydrogel having a crosslinked network structurewhich inhibits the penetration thereinto of bacteria, fungi, and/orregenerated tissue of the plant.

Further, in the above support, the network structure material in a driedstate has a dimension in the range of 0.1 μm to 1 cm, and has a carriershape of either of micro-beads, fibers, film, or indeterminate shape.

The present invention further provides a method of growing orregenerating a plant, wherein a carrier comprising a culture medium anda polymer constituting a network structure is used so as to grow orregenerate a plant while suppressing: the propagation of bacteria andfungi; the culture medium being substantially absorbed into and retainedby the network structure in a proportion of 10% to 100% of theequilibrium culture medium absorption of the polymer constituting thenetwork structure.

In the above support according to the present invention, in general, thenetwork structure comprising the above polymer has an ability to absorba culture medium so as to form a hydrogel. In other words, the carrieraccording to the present invention generally assumes a hydrogel state.

In the carrier according to the present invention, it is preferred touse a polymer which has been obtained by crosslinking a water-soluble orhydrophilic polymer compound, as the polymer constituting the networkstructure. This type of the polymer has a property such that it absorbswater to be swollen in an aqueous solution, but is not to dissolvedtherein. The equilibrium culture medium absorption (amount) as describedhereinafter-can be changed by changing the kind of the above-mentionedwater-soluble or hydrophilic polymer, and/or the degree of crosslinking.

When the above-mentioned support according to the first embodiment ofthe present invention is used as the above polymer constituting thenetwork structure, it is possible to effectively utilize the property ofthe hydrogel particles having a crosslinked structure and having adimension, in a dried state, in the range of 0.1 μm to 1 cm, that theyare swollen in water or a culture medium so as to reversibly increasethe volume thereof.

When a culture medium is completely absorbed into the above "supportaccording to the first embodiment" so as to constitute the carrieraccording to the present invention, the resultant "a state at which theculture medium is completely absorbed" is usually a semi-solid gelstate. Therefore, the resultant carrier may preferably be used as asupport for culturing a plant. In addition, it is also possible toeasily supplement such a gel with water, a nutrient, etc., which havebecome insufficient in the culturing process, as an additional (orsupplemental) fertilizer.

In addition, the plant which has been grown or regenerated by using thecarrier according to such an embodiment of the present invention mayeasily be collected or transferred, e.g., by adding an excess of wateror a culture medium to the carrier in the gel state so as to convert thegel into a sol state without damaging the resultant regenerated tissue.

On the other hand, when the above "support according to the secondembodiment" is used as the above polymer constituting the networkstructure, it is possible to effectively utilize a property of thepolymer comprising a crosslinked temperature-responsive polymer having aLCST, such that it absorbs water in water or a culture medium at atemperature lower than the LCST to be swollen; and it releases water tobe shrunk at a temperature higher than the LCST, thereby to markedlychange the volume thereof.

When the hydrogel particles comprising the crosslinkedtemperature-responsive polymer are used as the support according topresent invention, it is possible to easily remove a nutrient such asglucose required for the propagation of bacteria and fungi, from theinterior of the carrier only by using a temperature change, therebysuppressing the bacteria and fungi more effectively. Further, it is alsopossible that the culturing waste accumulated in the carrier is removedonly by utilizing the above temperature change, and the carrier is thenabsorbs a fresh culture medium and the resultant carrier is used for theculture process; or the resultant carrier is used after the transfer tofarm cultivation. Further, the conversion between the liquid state andthe gel state can be performed by utilizing a temperature change, andtherefore it is possible to transfer or collect the grown or regeneratedplant substantially without damaging the plant.

According to the investigations of the present inventor, the reason forthe above phenomenon, that the propagation of bacteria and fungi iseffectively suppressed by using the support or carrier according to thepresent invention, is presumed to be that the propagation of bacteriaand fungi is much faster, and the metabolic rate thereof is much larger,than those of a plant, therefore the necessity of the supplement ordelivery of a nutrient (such as water and saccharide) from the culturemedium to the bacteria and fungi is much greater than that in the planttissue.

More specifically, according to the investigations of the presentinventor, it is in the carrier according to the present invention havingthe above-mentioned structure, the culture medium which has beenabsorbed in the network structure comprising the polymer is hardlyavailable to the bacteria and fungi having a high metabolic activity,unlike a usual liquid medium, on the other hand such a culture medium isavailable to a plant tissue having a low metabolic activity,substantially equally in the case of the usual liquid medium, wherebythe propagation of bacteria and fungi is effectively and substantiallysuppressed without affecting the growth or regeneration of the plant.

The above-mentioned support or carrier according to the presentinvention may be used under a sterilized condition (e.g., in asterilized culture vessel), and may also be used under a non-sterilizedcondition as described hereinafter. The "culture under a non-sterilizedcondition" refers to a culture or cultivation process in an open systemother than a culture process in a sterilized closed system. Specificexamples of the "cultivation or culture under a non-sterilizedcondition" includes, e.g., cultivation in an open-air system such asgreenhouse, vinyl house, field and farm, and culture in a vessel under anon-sterilized condition.

Under a non-sterilized environment, it is possible to use the support orcarrier according to the present invention in combination with anothersupport or carrier (such as soil) as desired. In other words, it ispossible to use the support or carrier according to the presentinvention in a mixture with "another support or carrier". Further, atthe time of transfer or repotting, etc., it is also possible that aplant carrying the support or carrier according to the present inventionattached thereto is transferred to the culture or cultivation thereofusing "another support or carrier" under a non-sterilized condition.

At the time of such transfer of a plant from a sterilized condition to anon-sterilized condition, as described in Examples appearinghereinafter, the support or carrier per se, and/or a culture medium ornutrient (such as saccharide) may be removed from the plant as desired,and thereafter the resultant plant is transferred to the non-sterilizedcondition. If a factor capable of inhibiting the culture or cultivationunder the non-sterilized condition (such as saccharide and culturemedium to be used under the sterilized condition) can be removed, theremoval of the carrier or support per se from the plant is omissible.That is, the resultant plant may be transferred to another carrier suchas soil, while the carrier or support is left being attached to theroot, etc., of the plant.

Regardless of the sterilized or non-sterilized condition, when thesupport or carrier according to the present invention (especially,support or carrier comprising a polymer having a LCST) is used as atleast a part of a support or carrier for growing a plant, the support orcarrier may further contain another substance other than the cultureliquid medium (such as natural organic and inorganic substance) mixedtherein. When "another substance" is mixed in the support or carrier,the possibility of propagation of various germs tends to be somewhathigher because of the presence of voids or carbon sources, but on thebasis of the feature of the support or carrier according to the presentinvention, it is possible to supplement a nutrient or fertilizer, theremoval of a culturing waste including used or old culture medium, etc.,without damaging the plant. Accordingly, even when the carrier orsupport contains "another substance" mixed therein, the use of thesupport or carrier according to the present invention is effective forthe growth of a plant.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing an example of the temperature-dependentparticle size change in the micro-bead type carrier according to thepresent invention.

FIG. 2 is a graph showing an example of the temperature-dependent volumechange of a micro-bead type carrier according to the present invention.

FIG. 3 (Table 1) is a table showing the data corresponding to the aboveFIGS. 1 and 2.

FIG. 4 (Table 2) is a table showing the equilibrium water absorptiondata of a polymer support used in Example appearing hereinafter.

FIG. 5 (Table 3) is a table showing the composition of a liquid mediumused in Example appearing hereinafter.

FIG. 6 (Table 4) is a table showing the results of the growth obtainedunder a non-sterilized condition in Example appearing hereinafter.

DETAILED DESCRIPTION

Hereinbelow, the present invention will be described in detail withreference to the accompanying drawings, as desired.

(Equilibrium culture medium absorption)

In the carrier according to the present invention, it is preferred thata culture medium is absorbed into and retained in the network structurecomprising a polymer, in a proportion of 10%-100% of the equilibriumculture medium absorption. Herein, the "equilibrium culture mediumabsorption (E_(a))" is defined as the weight of a culture medium whichhas been absorbed into a polymer to be used for the carrier according tothe present invention, when the polymer (in a dried state, weight W_(p)=1 g) is immersed in a large excess of the culture medium (culturemedium to be used for the carrier according to the present invention) ata predetermined temperature (about 25° C.) for at least 3 days, untilthe swelling of the polymer reaches an equilibrium. With respect to this"equilibrium of swelling", e.g., a paper of T. Tanaka, et al., Phys.Rev. Lett., 55, 2455 (1985) may be referred to.

More specifically, when the total weight of (polymer)+(culture mediumabsorbed by polymer) after the swelling of the polymer reaches anequilibrium is represented by W_(e), the equilibrium culture mediumabsorption (E_(a))=W_(e) -W_(p).

In the present invention, the state at which the polymer has completelyabsorbed the culture medium is a state such that the ratio (M/G) of theweight of the polymer (G) to the weight of the culture medium (M) is notlarger than the equilibrium culture medium absorption E_(a).

Accordingly, in view of the suppression of the propagation of bacteriaand fungi in the carrier according to the present invention, it ispreferred that the above M/G ratio is not larger than the equilibriumculture medium absorption E_(a) of the polymer (i.e., M/G≦E_(a)). As theabove M/G ratio becomes smaller, the effect of the suppression of thepropagation of the bacteria and fungi is enhanced. However, when the M/Gratio becomes too small, it is possible that the ability of the carrierto absorb the culture medium exceeds the ability of the plant tissue toabsorb the culture medium, whereby the supplement of the nutrient suchas water to the plant tissue possibly becomes difficult.

From such a viewpoint, there is a region wherein the M/G ratio has apreferred value. Such a "preferred region" can be different depending onthe kind of a plant to be grown, or the kind of the bacteria or Fungi tobe prevented, but in general, the M/G ratio in the carrier according tothe present invention may preferably be 10%-100%, more preferably20%-60% of the equilibrium culture medium absorption E_(a) of thepolymer constituting the carrier.

As described above, the conventional liquid culture process or cultureprocess using an agar gel (a culture medium, etc., is present even invoids provided between the plant and the agar gel, and therefore such aculture process is equivalent to the liquid culture processmicroscopically or substantially) provides an environment which can be ahotbed for the propagation of bacteria and fungi. On the contrary, whenthe carrier according to the present invention is used, the liquidmedium is not substantially present in the carrier, and therefore thepropagation of bacteria and fungi is effectively suppressed.

(Shape of carrier)

The shape or form of the carrier according to the present invention isnot particularly limited, but may appropriately be selected depending onthe kind or dimension of a plant to be grown or regenerated. Specificexamples of the shape of the carrier may include various shapes such asparticles, micro-beads, fibers, flakes, sponge-like shape, film-likeshape, and sheet-like (indeterminate) shape.

The dimension of the network structure material in a dried statecomprising a polymer constituting the carrier according to the presentinvention can appropriately be selected depending on the kind or size ofplant tissue to be grown or regenerated, etc., dimension may generallybe in the range of 0.1 μm to 1 cm, more preferably in the range of 1 μmto 1 mm.

In the carrier according to the present invention, the above-mentioned"dimension in a dried state" refers to the average of maximum diameters(maximum dimensions) of the particles of the network structure materialconstituting the carrier (average of values obtained by measuring atleast 10 particles). More specifically, for example, the following sizemay be treated as the "dimension in a dried state" in accordance withthe shape of the above particles.

Micro-bead shape: particle size (average particle size)

Fiber shape: average of lengths of respective fiber pieces

Film shape, indeterminate shape: average of maximum dimensions ofrespective pieces

In the present invention, in place of the above "average of maximumvalues", it is also possible to use the diameter of a "ball" having avolume equal to the average of the volumes of respective pieces (averageof values obtained by measuring at least 10 pieces) as the "dimension ina dried state" of the particles of the above network structure material.

In an embodiment wherein the carrier according to the present inventionis shaped into the above particle form, when the regenerated planttissue penetrates into the carrier which has absorbed a culture medium,the regenerated plant tissue can penetrate into the carrier so as to begrown along gaps or clearances between the carrier particles, withoutdestroying the gel, unlike in the case of the conventional agar gel.

Further, the culture medium in a liquid state is not present in thevoids between the carrier particles which have been provided by theregenerated tissue having been grown and penetrated into the carrierparticles, as described hereinabove, and the carrier particle which hasabsorbed the culture medium generally becomes a very flexible hydrogel,whereby the voids may be mechanically filled with the hydrogel.

As described above, when a carrier in a particle form is used, unlike inthe case of the conventional agar gel, etc., the space which has beenformed due to the penetration of the regenerated tissue of a plant intothe carrier is promptly sealed, but also the liquid culture mediumcapable of being a hotbed of the propagation of bacteria and fungi isalso absorbed into the interior of the carrier. Accordingly, even whenbacteria and fungi are temporarily attached to the surface of thecarrier, unlike in the case of using the conventional agar gel, there isno problem such that bacteria and fungi penetrate into the gel to bepropagated through the voids which have been formed between theregenerated tissue and the agar gel.

Hereinbelow, there will be explained in detail the respective componentsconstituting the support or carrier according to the present invention.

(Culture medium)

In the present invention, as the culture medium or culturing liquid tobe used in combination with the above-mentioned polymer, it is possibleto use a known culture medium or culturing liquid (for growing and/orregenerating a plant) containing substantially no agar such as aMurashige-Skoog culture medium (MS-Culture Medium) without particularlimitation.

(Water-soluble or hydrophilic polymer)

Specific examples of the water-soluble or hydrophilic polymerconstituting a network structure or hydrogel constituting the carrieraccording to the present invention may include: methyl cellulose,dextran, polyethylene oxide, polypropylene oxide, polyvinyl alcohol,poly N-vinyl pyrrolidone, polyvinyl pyridine, polyacrylamide,poly-N-methyl acrylamide, poly-N-isopropyl acrylamide, poly-N,N-diethylacrylamide, poly-N-cyclopropyl acrylamide, poly-N-acryloyl pyrrolidine,poly-N,N-ethyl methyl acrylamide, poly-N-ethyl acrylamide,polymethacrylamide, poly-N-n-propyl methacrylamide, poly-N-isopropylmethacrylamide, poly-N-cyclopropyl methacrylamide, polyhydroxyethylacrylate, polyhydroxymethyl acrylate, polyacrylic acid, polymethacrylicacid, polyvinylsulfonic acid, polystyrenesulfonic acid and their salts,poly-N,N-dimethylaminoethyl methacrylate, poly-N,N-diethyl aminoethylmethacrylate, poly-N,N-dimethylaminopropyl acrylamide, and their salts,etc.

(Crosslinked structure)

As a method of imparting a crosslinked structure to the above polymer,there are a method of introducing a crosslinked structure at the time ofthe polymerization of a monomer; and a method of introducing acrosslinked structure after the completion of polymerization of amonomer. In the present invention, either of these methods may beadopted.

The former method may preferably be conducted by copolymerizing amonomer for providing the above water-soluble or hydrophilic polymer,and a bifunctional monomer (or multi-functional monomer having at leasttwo functional groups). Specific examples of such a bifunctional monomermay include: N,N-methylenebis-acrylamide, hydroxyethyl dimethacrylate,divinylbenzene, etc.

In the latter method, it is typical form a crosslink between moleculesby utilizing the energy such as light, electron beam, and γ-rayirradiation.

Alternatively, the latter method can also be conducted by crosslinkingthe above water-soluble or hydrophilic polymer by using, as acrosslinking agent, a multi-functional molecule having therein aplurality of functional groups (such as isocyanate group) which iscapable of being bonded to a functional group (such as amino group) ofthe above water-soluble or hydrophilic polymer.

In the present invention, the above-mentioned "equilibrium culturemedium absorption" of a polymer for providing the network structure isdependent on the crosslinked structure, crosslinking density (or densityof crosslinking) of the polymer. As the crosslinking density becomeslower, the equilibrium culture medium absorption is increased. In theformer method (wherein the crosslinked structure is introduced at thetime of the polymerization of a monomer), the crosslinking density canarbitrarily be controlled, e.g., by changing the copolymerization ratioof the bifunctional monomer. In the latter method (wherein thecrosslinked structure is introduced after the completion of thepolymerization of a monomer), the crosslinking density can arbitrarilybe controlled, e.g., by changing the quantity of irradiation such aslight, electron beam, and γ-ray.

In the present invention, the crosslinking density may preferably be inthe range of 0.2 mol % to 10 mol %, more preferably 0.5 mol % to 4 mol%, in terms of the ratio of the moles of the branching point to themoles of all the monomer. Alternatively, when the crosslinked structureis introduced by the former method, the crosslinking density maypreferably be in the range of 0.3 wt % to 3 wt %, more preferably 0.5 wt% to 2 wt %, in terms of the copolymerization weight ratio of thebifunctional monomer to all the monomers (inclusive of the bifunctionalmonomer per se).

When the crosslinking density exceeds the above-mentioned range thereof,the culture medium-absorbing ability of the carrier according to thepresent invention is reduced, the effect of the carrier for suppressingthe propagation of bacteria and fungi is reduced, and simultaneously theamount of the culture medium contained in the carrier is reduced and theability thereof for supplementing a nutrient such as water to a plant isalso reduced. On the other hand, when the crosslinking density is belowthe above-mentioned range, the network structure of the hydrogel becomessparse, bacteria, Fungi or regenerated tissue is more liable topenetrate into the gel, and simultaneously the gel becomes mechanicallyweak, and is less able to function as a support for culturing a plant.

The crosslinking density (molar ratio of the branching points withrespect to all the monomer) may be determined, e.g., by ¹³ C-NMR(nuclear magnetic resonance absorption) measurement, IR (infraredabsorption spectrum) measurement, or elemental analysis.

(Shaping method)

As the method of shaping of the carrier according to the presentinvention, it is possible to use an ordinary method of shaping apolymer.

When the simplest method is used, a monomer for providing thewater-soluble or hydrophilic polymer, the above-mentioned bifunctionalmonomer, and a polymerization initiator are dissolved in water, andsubjected to polymerization by use of heat or light, whereby a hydrogelcan be prepared. The resultant hydrogel is mechanically crushed orpulverized, the unreacted monomer, the remaining polymerizationinitiator, etc., are removed therefrom by washing with water, andthereafter the resultant product is dried, thereby to provide a carrieraccording to the present invention.

Further, when the monomer for providing the water-soluble or hydrophilicpolymer is liquid, the bifunctional monomer and polymerization initiatorare added into the monomer, the resultant mixture is polymerized by bulkpolymerization by use of heat or light, the resultant product ismechanically crushed, the unreacted monomer and the remainingbifunctional monomer are removed therefrom by extraction with water, andthe product is dried, whereby a carrier according to the presentinvention can be provided.

On the other hand, when the carrier according to the present inventionin a micro-bead form is intended to be prepared, it is possible to usean emulsion polymerization method, a suspension polymerization method, aprecipitation polymerization method, etc. Particularly, a reverse-phasesuspension polymerization method may preferably be used. In thereverse-phase suspension polymerization method, as a dispersion medium,an organic solvent which doesn't dissolve the monomer and the resultantpolymer is preferred. For example, a saturated hydrocarbon such ashexane is preferred. In addition, it is also possible to use asurfactant (e.g., a nonionic surfactant such as sorbitan fatty acidester) as a suspension auxiliary in combination with the above organicsolvent.

The particle size of the resultant micro-bead can be controlled by thekind or amount of the surfactant to be added, the stirring speed, etc.As the polymerization initiator, either of a water-solublepolymerization initiator, and a water-insoluble polymerization initiatorcan be used.

When the carrier according to the present invention is formed into afiber shape, film shape, etc., for example, it is possible to use amethod wherein an aqueous solution of a water-soluble polymer isextruded into an organic solvent which is unmixable with water by usinga die, etc., to form each of the predetermined shapes, then theresultant product is irradiated with light, electron beam, γ-ray, etc.,so as to impart a crosslinked structure to the polymer. Further, it isalso possible to use a method wherein the above water-soluble polymer isdissolved in an organic solvent or water, is shaped by a solvent castingmethod, and then is irradiated with light, electron beam, γ-ray, etc.,so as to impart a crosslinked structure to the polymer.

Further, it is also possible to mechanically crush either of theresultant shaped products having various shapes obtained by the abovemethods so as to shape the product into a support having a desireddimension.

(Liquid-gel transition temperature)

In the present invention, the terms "liquid state" "gel state" and"liquid-gel transition temperature" are defined in the following manner.With respect to these definitions, a paper (Polymer Journal, 18(5),411-416 (1986)) may be referred to.

Thus, 1 mL of a dispersion (liquid) of a support is poured into a testtube having an inside diameter of 1 cm, and is left standing for 12hours in a water bath which is controlled at a predetermined (constant)temperature. Thereafter, in a case where the interface (meniscus)between the dispersion of the support and air is deformed (inclusive acase wherein the dispersion of the support flows out from the test tube)due to the weight of the dispersion of the support per se when the testtube is turned upside down, the above dispersion of the support isdefined as a "liquid state" at the above-mentioned predeterminedtemperature.

On the other hand, in a case where the interface (meniscus) between thedispersion of the support and air is not deformed due to the weight ofthe dispersion of the support per se even when the test tube is turnedupside down, the above dispersion of the support is defined as a "gelstate" at the above-mentioned predetermined temperature.

On the other hand, when the temperature at which the "gel state" isconverted into the "liquid state" is determined while graduallyincreasing the above "predetermined temperature" (e.g., in 1° C.increment), the thus determined transition temperature is defined as a"liquid-gel transition temperature". At this time, alternatively, it isalso possible to determine the above temperature at which the "liquidstate" is converted into the "gel state" while gradually decreasing the"predetermined temperature" (e.g., in 1° C. decrement).

In the present invention, the above liquid-gel transition temperaturemay preferably be higher than 0° C. and not higher than 60° C., morepreferably, not lower than 4° C. and not higher than 50° C.(particularly preferably, not lower than 4° C. and not higher than 40°C.), in view of the prevention of thermal damage to a plant. The polymerhaving such a preferred liquid-gel transition temperature may easily beselected from specific compounds as described below, according to theabove-mentioned screening method (method of measuring the liquid-geltransition temperature).

In the process for growing or regenerating a plant according to thepresent invention, it is preferred to set the above-mentioned liquid-geltransition temperature (a °C.) between the temperature at which theplant is grown or regenerated (b °C.), and the temperature at which thegrown or regenerated plant is collected or transferred (c °C.). In otherwords, the above-mentioned three kinds of temperatures of a °C., b °C.and c °C. may preferably have a relationship of b<a<c. Morespecifically, the value of (a-b) may preferably be 1°-40° C., morepreferably 2°-30° C. On the other hand, the value of (c-a) maypreferably be 1°-40° C., more preferably 2°-30° C.

The temperature-responsive polymer having a LCST to be used in thepresent invention has a property such that the polymer is insoluble inwater or a culture medium at a temperature higher than the LCST, but isconverted into a soluble state at a temperature lower than the LCST.

It is considered that the state change of the temperature-responsivepolymer is based on the hydration and dehydration phenomena. Withrespect to such phenomena, Haskins, M., et al.; J. Macromol. Sci. Chem.,A2(8), 1441, 1968, provides a description thereof by usingpoly-N-isopropyl acrylamide (PNIPAAm) as an example of such polymers.The PNIPAAm is a polymer having a negative solubility-temperaturecoefficient with respect to water. At a lower temperature, there isformed a hydrate (oxonium hydroxide) depending on the hydrogen bondingbetween the PNIPAAm molecule and the water molecule. However, it isconsidered that the hydrate is decomposed by increasing the temperatureup to a value higher than the LCST to be dehydrated, and as a result,PNIPAAm molecules are aggregated with each other to be precipitated.

When a crosslinked structure is imparted to the abovetemperature-responsive polymer having a LCST, the resultant polymer isnot dissolved but can retain a swollen gel state even in water or aculture medium at a temperature lower than the LCST. On the other hand,when the temperature is raised to a value higher than the LCST, thepolymer is converted into a water-soluble state, whereby water isseparated from the gel and the volume of the crosslinked material ismarkedly decreased. As described above, the support for growing orregenerating a plant and the method for growing or regenerating a plantaccording to the present invention utilize such a property of thecrosslinked temperature-responsive polymer.

(Temperature-responsive polymer)

Preferred examples of the temperature-responsive polymer to be used inthe present invention may include: e.g., poly N-substituted acrylamidederivative, poly N-substituted methacrylamide derivative, and thesecopolymers; polyvinyl methyl ether, polypropylene oxide, polyethyleneoxide, etherified methyl cellulose, partially acetylated polyvinylalcohol, etc. Particularly preferred examples thereof to be used in thepresent invention may include: poly N-substituted acrylamide derivativeor poly N-substituted methacrylamide derivative or these copolymers,polyvinyl methyl ether, polypropylene oxide, partially acetylatedpolyvinyl alcohol.

Preferred examples of the polymer to be used in the present invention isexemplified below in a sequence of from one having a lower LCST to onehaving a higher LCST:

poly-N-acryloyl piperidine;

poly-N-n-propyl methacrylamide;

poly-N-isopropyl acrylamide;

poly-N,N-diethyl acrylamide;

poly-N-isopropyl methacrylamide;

poly-N-cyclopropyl acrylamide;

poly-N-acryloyl pyrrolidine;

poly-N,N-ethyl methyl acrylamide;

poly-N-cyclopropyl methacrylamide;

poly-N-ethyl acrylamide

The above polymer may be either a homopolymer or a copolymer comprisinga monomer constituting the above polymer and "another monomer". The"another monomer" to be used for such a purpose may be either ahydrophilic monomer, or a hydrophobic monomer.

Specific examples of the above hydrophilic monomer may include: N-vinylpyrrolidone, vinyl pyridine, acrylamide, methacrylamide, N-methylacrylamide, hydroxyethyl methacrylate, hydroxyethyl acrylate,hydroxymethyl methacrylate, hydroxymethyl acrylate, methacrylic acid andacrylic acid having an acidic group, and salts of these acids,vinylsulfonic acid, styrenesulfonic acid, etc., and derivatives having abasic group such as N,N-dimethylaminoethyl methacrylate,N,N-diethylaminoethyl methacrylate, N,N-dimethylaminopropyl acrylamide,salts of these derivatives, etc. However, the hydrophilic monomer to beusable in the present invention is not restricted to these specificexamples.

On the other hand, specific examples of the above hydrophobic monomermay include: acrylate derivatives and methacrylate derivatives such asethyl acrylate, methyl methacrylate, butyl methacrylate, and glycidylmethacrylate; N-substituted alkyl methacrylamide derivatives such asN-n-butyl methacrylamide; vinyl chloride, acrylonitrile, styrene, vinylacetate, etc. However, the hydrophobic monomer to be usable in thepresent invention is not restricted to these specific examples.

In general, when the above temperature-responsive polymer iscopolymerized with a hydrophilic monomer, the resultant LCST may beincreased. On the other hand, when the above temperature-responsivepolymer is copolymerized with a hydrophobic monomer, the resultant LCSTmay be decreased. Accordingly, the liquid-gel transition temperature ofa dispersion of the temperature-responsive polymer to which acrosslinked structure has been imparted according to the presentinvention is increased by using a temperature-responsive polymer whichhas been copolymerized with a hydrophilic monomer, and the liquid-geltransition temperature is decreased by using a temperature-responsivepolymer which has been copolymerized with a hydrophobic monomer.Accordingly, it is also possible to control the liquid-gel transitiontemperature in the present invention by selecting the monomer componentto be used for such copolymerization.

As the method of imparting a crosslinked structure to atemperature-responsive polymer, there are a method wherein a crosslinkedstructure is introduced into the polymer at the time of thepolymerization of the monomer for providing the temperature-responsivepolymer; and a method wherein a crosslinked structure is introduced to atemperature-responsive polymer after the completion of thepolymerization of the monomer. In the present invention, however, it ispossible to adopt each of these methods.

The former method can generally be conducted by utilizing thecopolymerization with a bifunctional monomer (or a monomer having threeor more functional groups). For example, such a method may be conductedby using a bifunctional monomer such as N,N-methylene bis-acrylamide,hydroxyethyl dimethacrylate, and divinylbenzene.

The latter method can generally be conducted by forming a crosslinkbetween molecules by utilizing light, electron beam, γ-ray irradiation,etc.

Further, the latter method may also be conducted by crosslinking atemperature-responsive polymer, e.g., by using, as a crosslinking agent,a multi-functional molecule having a plurality of functional group (suchas isocyanate group) which is capable of being bonded to a functionalgroup (such as amino group) in the temperature-responsive polymer.

(Ratio of volume change)

With respect to the ratio (magnification) of the volume change in thesupport according to the present invention in water, when the volume ofthe shrunk support at a temperature higher than the LCST is defined as"1" (one), the equilibrium swelling volume of the support at atemperature for growing or regenerating a plant, which is lower than theLCST, may preferably be 1.1-100, more preferably 5-100. Herein, the"equilibrium swelling volume" refers to the volume which has beenprovided after the support according to the present invention is soakedin an excess of water at a predetermined temperature (constanttemperature) for at least three days so that the swelling thereofreaches an equilibrium. With respect to such an equilibrium swellingvolume, e.g., a paper of T. Tanaka, et al., Phys. Rev. Lett. 55, 2455(1985) may be referred to.

The above-mentioned ratio of the temperature-dependent volume change ofthe support according to the present invention is generally dependent onthe crosslinked structure thereof, particularly the crosslinkingdensity, and has a tendency such that as the crosslinking densitybecomes lower, the volume change becomes larger. The crosslinkingdensity can arbitrarily be controlled, e.g., by changing thecopolymerization ratio of the bifunctional monomer in the former method,and e.g., by changing the quantity of irradiation with light, electronbeam, γ-ray, etc., in the latter method.

The preferred range of the crosslinking density may be about 0.2 mol %to about 10 mol %, more preferably about 0.5 mol % to about 4 mol %, interms of the ratio of the moles of branching points to the moles of allthe monomer. When the crosslinked structure is introduced by using theformer method, the copolymerization weight ratio of the bifunctionalmonomer to that of all the monomers (inclusive of the bifunctionalmonomer per se) may preferably be in the range of about 0.3 wt. % toabout 3 wt. % (more preferably about 0.5 wt. % to about 1.5 wt. %).

In the present invention, when the crosslinking density exceeds theabove-mentioned range of about 0.2 mol % to about 10 mol %, the ratio ofthe temperature-dependent volume change in the support according to thepresent invention is reduced, whereby clear liquid-gel conversion isless likely to occur. On the other hand, when the crosslinking densityis below the above-mentioned range, the mechanical strength of thesupport according to the present invention is reduced, and thedispersion thereof is less likely to retain a strength enough to supporta plant in a gel state thereof.

(Dimension and manufacturing method for support)

The dimension of the-support according to the present invention canappropriately be selected depending on the kind and dimension of a plantto be grown or regenerated. When the support has a particle shape or amicro-bead shape, the support may preferably have a particle size in therange of 0.1 μm to 1 cm (more preferably 1 μm to 1 mm) at the time ofthe shrinkage of the support in an aqueous dispersion, i.e., at atemperature higher than the LCST of the temperature-responsive polymerconstituting the support.

Particularly, when the support has a particle shape or a micro-beadshape, it is preferred to use an emulsion polymerization method, asuspension polymerization method, a precipitation polymerization method,etc. As the method of imparting a crosslinked structure to thetemperature-responsive polymer, it is possible to use a method whereincrosslinking is effected by using a bifunctional monomer at the time ofthe polymerization of a monomer; a method wherein the polymerization ofa monomer is completed and the resultant product is shaped, andthereafter crosslinking is effected by using light, electron beam, γ-rayirradiation, etc., as described hereinabove.

Particularly, a reverse-phase suspension polymerization method maypreferably be used, when the crosslinked temperature-responsive polymerin the form of micro-beads is synthesized from a water-soluble monomerand a water-soluble bifunctional monomer.

In the reverse-phase suspension polymerization method, it is preferredto use, as a dispersion medium, an organic solvent which does notdissolve the monomer and the produced polymer. For example, a saturatedhydrocarbon such as hexane may preferably be used. Further, it is alsopossible to use a surfactant (e.g., a nonionic surfactant such assorbitan fatty acid ester) as a suspension auxiliary in combination withthe above organic solvent. The particle size of the micro-bead to beobtained can be controlled by the kind or amount of the surfactant to beadded, the stirring speed, etc. As the polymerization initiator, it ispossible to use either of a water-soluble polymerization initiator, andwater-insoluble polymerization initiator. In view of effectivecollection or recovery of the polymerization product, it is preferred toperform the polymerization at a temperature lower than the LCST of theabove temperature-responsive polymer, and therefore a low-temperaturepolymerization initiator such as redox polymerization initiator maypreferably be used.

When the support according to the present invention is formed into afiber shape, a flake shape, a sponge shape, a particle shape, etc., forexample, it is possible to use a method wherein an aqueous solution of atemperature-responsive polymer which has been cooled to a temperaturelower than the LCST thereof is extruded into water at a temperaturehigher than the LCST, or into an organic solvent which is unmixable withwater by using a die. When such a shaping method is used, a crosslinkedstructure may be imparted to the polymer by using irradiation withlight, electron beam, γ-ray, etc.

When the support according to the present invention is formed into aplate shape or a film shape, for example, it is possible to use a methodwherein the above temperature-responsive polymer is dissolved in anorganic solvent or water at a temperature lower than the LCST, and isshaped by a solvent casting method. When such a shaping method is used,a crosslinked structure may also be imparted to the polymer by usingirradiation with light, electron beam, γ-ray, etc.

Further, it is also possible to mechanically crush either of theresultant shaped products having various shapes obtained by the abovemethods so as to shape the product into a support having a desireddimension.

In the present invention, it is possible to appropriately select thedispersion concentration, in a culture medium for culturing a plant, ofthe support comprising the temperature-responsive polymer having theabove crosslinked structure, depending on the kind and shape of theplant. The concentration, however, may generally be 0.1-30 wt. %, morepreferably 1-10 wt. %.

(Process for growing/regenerating plant)

The application of the polymer support or carrier according to thepresent invention to the process for growing or regenerating a plant isnot particularly limited, but the carrier according to the presentinvention is particularly effectively usable in the application of theroot-originating step of a plant. Hereinbelow, there is described anexample of such a root-originating step of a plant.

Thus, a polymer for providing a network structure (e.g., in aparticulate shape) is added into a desired culture medium forconstituting a carrier according to the present invention so as toprovide an M/G ratio which is not larger than the equilibrium culturemedium absorption, and the polymer is caused to completely absorb theculture medium, thereby to cause the polymer to form a gel state. Thedispersion concentration of the polymer support in the culture mediummay appropriately be selected depending on the kind of the culturemedium, and the kind, shape, dimension, etc., but in general, theconcentration may preferably be 0.1-30 wt. %, more preferably 1.0-10 wt.%.

Subsequently, an organ or seedling of a plant such as leaf, stem, root,petal, and anther (pollen) is disposed on or inserted into the abovegel, and is cultured under a condition under which the gel state isretained, thereby to originate a root in the gel. At this time, asdescribed hereinabove, the regenerated root is grown while it pushes thecarrier particles apart, and the regenerated root is completely shieldedor screened from the external environment by the carrier, and thereforethe propagation of bacteria and fungi is suppressed.

(Growth or regeneration of plant using support according to firstembodiment)

Hereinbelow, there is described a preferred example of the process forgrowing or regenerating a plant by using the above support according tothe first embodiment.

First of all, the above-mentioned polymer support is uniformly dispersedin a desired culture medium. Then, into the resultant culture mediumdispersion of the polymer support, a plant organ such as leaf, stem,root, petal, anther (pollen), and seedling, or a plant tissue such ascallus, hair root and protoplast which has been regenerated from theabove plant is mixed and dispersed. The support absorbs the culturemedium, and the volume thereof is increased, and the fluidity thereof ismarkedly reduced so as to be converted into a gel state. Accordingly, itis possible that the plant is supported in the gel and the plant isgrown or regenerated therein.

On the other hand, the collection (or recovery) or transfer of the plantwhich has been grown or regenerated in the gel may easily be effected byadding an excess of water or a culture medium (water or culture mediumin an amount of preferably 1.1-100 times, more preferably 1.5-10 timesin terms of weight ratio based on the weight of the swollen gel) ontothe gel of the support so as to again disperse the support to beliquidized. Particularly, when the root-originating plant is collectedor transferred, the root-originating plant may easily be separatedwithout damaging the plant. Herein, in the case of the polymer supportaccording to the present invention, the liquid-gel conversion canappropriately be controlled as described above, and this feature isimportant as compared with the agar gel as described hereinabove.

Conventionally, the operation for separating the plant after thecompletion of the root-originating stage thereof from the agar gel hasbeen a primary cause for the reduction in the efficiency of active rootanchoring of the plant after such an operation. However, as describedabove, the polymer support according to the present invention cancompletely solve such problems.

In addition, the supplement of a nutrient which becomes insufficient inthe above gel culture medium, or the removal of a waste material capableof inhibiting the growth or regeneration can also be performed byliquidizing the old gel to be removed in the above manner, and thentransferring the plant into a fresh culture medium. In the presentinvention, the gel culturing may easily be continued in such a manner.

Further, in the case of the above support, after the plant is grown orregenerated and then collected or transferred, the support may easily berecovered from the dispersion liquid of the support (by a method such ascentrifugal separation), the support is washed, whereby the resultantsupport can be recycled.

(Growth or regeneration of plant using support according to secondembodiment)

Hereinbelow, there is described an example of the process for growing orregenerating a plant by using the above-mentioned(temperature-responsive) support according to the second embodiment.

When the temperature-responsive polymer is used as a support, thepolymer support is uniformly dispersed at a temperature higher than theliquid-gel transition temperature of the support, and then thetemperature is decreased to a value lower than the liquid-gel transitiontemperature, whereby uniform gel is formed on the basis of the waterabsorption and swelling of the polymer. Onto the resultant gel, an organor seedling of a plant such as leaf, stem, root, petal, and anther(pollen) is disposed or inserted, and the plant is cultured whileretaining the gel state and the plant is caused to originate a root inthe gel. When the root-originating plant is collected or transferredfrom the gel, the gel is liquidized by raising the temperature to avalue higher than the liquid-gel transition temperature so as to causethe dehydration and shrinkage of the crosslinked polymer, and then theroot-originating plant may easily be separated from the dispersion ofthe shrunk carrier without damaging the plant. Herein, in the case ofthe polymer support according to the present invention, the liquid-gelconversion can appropriately be controlled as described above, and thisfeature is important as compared with the agar gel as describedhereinabove.

Further, at a temperature higher than the LCST, the above polymer is ina dehydrated and shrunk state, and therefore the viscosity of theculture medium dispersion containing the support comprising the polymeris substantially equivalent to the viscosity of the culture medium perse. Due to such a characteristic, not only the collection and transferof the plant are facilitated, but also the removal of the culture mediumby washing at the time of the collection and transfer is simplified, andfurther, the collection and transfer operations do not damage the plantat all.

In addition, the supplement of a nutrient which becomes insufficient inthe above gel culture medium, or the removal of a waste material capableof inhibiting the growth or regeneration can also be performed byliquidizing the gel at a temperature higher than the LCST to be removedtogether with the old culture medium. If a fresh culture medium is addedto the culture medium, the gel culturing may easily be continued, oralternatively, the resultant plant as such may be transferred to thecultivation in a field or farm, whereby an epochal saving of labor inthe transferring operations can be accomplished.

Further, in the case of the above support, after the plant is grown orregenerated and then collected or transferred, the support may easilyrecovered selectively from the dispersion liquid thereof (by a methodsuch as centrifugal separation), the support is washed, whereby theresultant support can be recycled.

Hereinbelow, the present invention will be described in more detail withreference to Examples. However, it should be noted that the presentinvention is defined by Claims, but is not limited by the followingExamples.

EXAMPLES Example 1

(Synthesis of micro-bead support)

7.5 g of acrylamide, 0.1 g of N,N'-methylenebis-acrylamide, and 0.1 g ofammonium persulfate were dissolved in 100 mL (milliliter) of distilledwater. The resultant aqueous solution was added into a solution whichhad been obtained by dissolving 10 g of sorbitan mono-oleate (SPAN-80,mfd. by Kanto Kagaku K.K.) in 1000 mL of hexane, and the resultantmixture was vigorously stirred under a nitrogen stream so as to form asuspension. Thereafter, 3 mL of N,N,N',N'-tetramethyl ethylenediaminewas added to the resultant mixture, and subjected to polymerization for4 hours at room temperature. The resultant aqueous phase was separatedfrom the reaction mixture, and the polymerization product was washedwith 500 mL of hexane three times. Subsequently, 1000 mL of distilledwater was added to the polymerization product and then stirred, andthereafter the mixture was left standing so as to precipitate a carrierin the form of micro-beads, and the supernatant liquid was discarded.Such a water-washing operation was repeated three times, and then theproduct was dried under vacuum, thereby to obtain 7 g of a support (A)in the form of micro-beads according to the present invention.

When the above support in distilled water was observed with an opticalmicroscope, spherical particles having a diameter of about 20-200 μmwere observed.

Example 2

A piece of lateral bud tissue of carnation was cut out, and washed withdetergent and aqueduct water, and immersed in 70% aqueous ethanolsolution for 30 sec., and immersed in 1% aqueous sodium hypochloritesolution for 7 min., to be sterilized, and further was washed withsterilized distilled water several times. The thus obtained lateral budas an explant was planted on an MS-culture medium (Murashige-Skoogculture medium, mfd. by Cosmo-Bio Co.) containing 0.8% of agar, andcultured at 25° C. for two weeks so as to form a shoot, and then theshoot was cut into pieces thereof corresponding to the respective nodes.

Subsequently, 0.5 g of the micro-bead support (A) prepared in Example 1was dispersed in 10 mL of an MS-culture medium containing 0.1 mg/L of1-naphthalene acetic acid, and 10 g/L of sucrose in terms of therespective concentration, and poured into a tube made of glass(culturing vessel) having an inside diameter of 2 cm and a length of 10cm, and sterilized by an autoclave treatment (121° C., for 20 minutes),and simultaneously the above support was swollen with the aboveMS-culture medium and formed into a gel state.

On the thus obtained gel, the above-mentioned shoots of the carnationwhich had been cut into pieces corresponding to the respective nodeswere transferred. As a result, the shoots were well retained in theabove gel. Further, when culturing was conducted for three weeks in sucha state, the origination of roots in the gel was observed.

Then, when the thus obtained root-originating plant was collected fromthe culturing vessel for the purpose of acclimation (or acclimatization)thereof, 15 mL of water was added into the culture vessel containing theroot-originating plant. As a result, the above micro-bead support wasimmediately dispersed and the dispersion culture medium was convertedform the gel state to a liquid state, whereby the root-originating plantcould easily be taken out from the culture vessel. In theabove-mentioned collecting step, the support (A) attached to the rootscould easily be removed therefrom by washing with water, no damage tothe roots was observed, and the operations per se were very easy.

Comparative Example 1

An MS-culture medium-containing 8 g/L of agar, 0.1 mg/L of1-naphthaleneacetic acid, and 10 g/L of sucrose in terms of theirconcentrations was prepared by an ordinary method, and was thensterilized by an autoclave treatment. Subsequently, the culture mediumwas cooled to 23° C. to be formed into a gel state, and thereafter, theshoot of the carnation which had been cut into pieces corresponding toits respective nodes was transferred onto the thus obtained gel, and wassubjected to a culture process in the same manner as in Example 2 for 3weeks. After three weeks counted from the initiation of the cultureprocess, it was recognized that roots were originated in the gel.

In order to separate the root-originating plant from the gel, the plantwas taken out from the culture vessel under a state under which the gelwas attached to the roots, and then was mechanically shaken in water byan ordinary method. As a result, it was difficult to remove the gel fromthe hair-like roots, but some of the hair-like roots were broken off byan operation of removing the gel by use of hands. Further, the washingoperation using hands was very troublesome.

Example 3

(Synthesis of micro-bead support)

15 g of N-isopropyl acrylamide (NIPAAm), 0.1 g ofN,N'-methylenebis-acrylamide (Bis), and 0.1 g of ammonium persulfatewere-dissolved in 100 mL of distilled water. The resultant aqueoussolution was added into a solution which had been obtained by dissolving10 g of sorbitan mono-oleate (SPAN-80, mfd. by Kanto Kagaku K.K.) in1000 mL of hexane, and the resultant mixture was vigorously stirredunder a nitrogen stream so as to form a suspension.

Thereafter, 3 mL of N,N,N',N'-tetramethylethylene diamine was added tothe resultant mixture, and subjected to polymerization for 4 hours atroom temperature.

The resultant aqueous phase was separated from the reaction mixture, andthe polymerization product was washed with 500 mL of hexane three times.Subsequently, 1000 mL of distilled water was added to the polymerizationproduct, and was cooled to 4° C. Thereafter, the mixture was heated upto 40° C. so as to shrink the resultant carrier in the form ofmicro-beads, and the supernatant liquid was discarded. Such awater-washing operation was repeated three times, and then the productwas dried under vacuum, thereby to obtain a support (B) in the form ofmicro-beads according to the present invention.

The above procedure was repeated except for using 0.4 g of Bis, therebyto obtain a support (C) in the form of micro-beads according to thepresent invention.

The diameters of the-support (B) and (C) were measured under an opticalmicroscope at various temperatures. The thus obtained results are shownin the graph of FIG. 1. In addition, these diameters are converted intoratios of volume change (magnifications) in terms of the ratio of thevolume to the volume (=1 (one)) of the shrunk micro-bead. The thusobtained results are shown in the graph of FIG. 2. FIG. 3 (Table 1)shows data corresponding to those of the above FIGS. 1 and 2.

(Preparation of support dispersion)

5 g of the dried support (B) obtained by the above procedure wasdispersed in 100 mL of a Murashige-Skoog culture medium for a plant(MS-culture medium, mfd. by Cosmo-Bio K.K.) at 40° C. When the resultantdispersion was gradually cooled, the dispersion lost its fluidity atabout 31° C. to be formed into a gel state. When the product in the gelstate was gradually warmed, the gel again returned to a dispersionliquid having a fluidity at about 32° C.

Example 4

A piece of lateral bud tissue of carnation was cut out, and washed withdetergent and aqueduct water, and immersed in 70% aqueous ethanolsolution for 30 sec., and immersed in 1% sodium hypochlorite solutionfor 7 min., to be sterilized, and further washed with sterilizeddistilled water several times. The thus obtained lateral bud tissue asan explant was planted on an MS-culture medium containing 0.8% of agar,and cultured for two weeks so as to form a shoot, and then the shoot wascut into pieces thereof corresponding to the respective nodes.

Subsequently, 5 g of the micro-bead support (B) prepared in Example 3was dispersed in 100 mL of an MS-culture medium containing 0.1 mg/L of1-naphthalene acetic acid, and 10 g/L of sucrose in terms of therespective concentrations. The resultant dispersion medium wassterilized by an autoclave treatment (121° C., for 20 minutes).

The dispersion medium of the support (B) obtained above assumed a liquidstate at 37° C., but was formed into a complete gel state by loweringthe temperature to about 23° C. After the dispersion medium of thesupport (B) was formed into a complete gel state in such a manner, theabove-mentioned shoots of the carnation which had been cut into piecescorresponding to the respective nodes were transferred onto the thusobtained gel. When culturing was conducted for three weeks at about 23°C., the origination of roots in the gel was observed after three weekscounted from the initiation of the culturing.

Then, when the thus obtained root-originating plant was collected fromthe culturing vessel for the purpose of acclimation thereof, thetemperature of the culture vessel containing the root-originating plantwas raised up to about 37° C., the volume of the above micro-beadsupport was immediately reduced and the dispersion culture medium wasconverted form the gel state to a liquid state, whereby theroot-originating plant could easily be taken out from the culturevessel. In the above-mentioned collecting step, the support (B) attachedto the roots could easily be removed therefrom by washing with water, nodamage to the roots was observed, and the operations per se were veryeasy.

Comparative Example 2

An MS-culture medium containing 8 g/L of agar, 0.1 mg/L of1-naphthaleneacetic acid, and 10 g/L of sucrose in terms of theirconcentrations was prepared by an ordinary method, and was thensterilized by an autoclave treatment. Subsequently, the resultantculture medium was cooled to about 23° C. to be formed into a gel state,and thereafter, the shoot of the carnation which had been cut intopieces corresponding to its respective nodes was transferred onto thethus obtained gel, and was subjected to culturing in the same manner asin Example 4 at about 23° C. for 3 weeks. After three weeks counted fromthe initiation of the culturing, it was recognized that roots wereoriginated in the gel.

In order to separate the root-originating plant from the gel, the plantwas taken out from the culture vessel under a state under which the gelwas attached to the roots, and then was mechanically shaken in water byan ordinary method. As a result, it was difficult to remove the gelattached to the hair-like roots from these roots, but some of thehair-like roots were broken off by an operation of removing the gel byuse of hands. Further, the washing operation using hands was verytroublesome.

Example 5

A stem of tobacco (Nicotina glutinosa) which had been grown for four tofive months was cut so as to provide a piece thereof having a length of4-5 cm, and washed with detergent and aqueduct water, and immersed in70% aqueous ethanol solution for 30 sec., and immersed in 1% sodiumhypochlorite solution for 7 min., to be sterilized, and was furtherwashed with sterilized distilled water several times.

Then, the above stem was cut by using a sterilized razor blade so as toprovide a piece thereof having a length of about 5 mm. A cork borerhaving a diameter of about 3 mm was stuck to the resultant slice so asto collect a marrow tissue thereof, and then the marrow tissue wasfurther cut into pieces of about 2-3 mm. All of the above-mentionedoperations were conducted under sterilized conditions.

Subsequently, 5 g of the support (B) in the form of micro-beadsaccording to the present invention prepared in Example 3 was dispersedin 100 mL of a culture medium for tobacco cell culture containing noagar (Atsushi Hirai, et al., Seibutsukagaku Jikken-ho (Methods ofBiochemistry) 16, "Shokubutsu Saibo Ikushu Nyuumon (Introduction toHybridization of Plant Cells)", p. 15, (1969), published byGakkai-Shuppan Center) at 37° C., and was sterilized by an autoclavetreatment.

Several slices of the marrow tissue obtained by the above method weredispersed in the culture medium containing the above support in the formof micro-bead at 37° C. Thereafter, the temperature was lowered to about23° C. so as to convert the culture medium into a complete gel state,whereby the marrow tissue was embedded in the gel. When the resultantgel was subjected to culturing for 15 days under artificial lightirradiation condition of 1000-3000 luxes, good callus formation from therespective marrow tissue pieces was observed.

Then, in order to collect the callus which had been formed in the gel,the gel was heated up to 37° C. for about 3 minutes so as to dissolvethe gel, and thereafter the resultant mixture was moderately centrifugedso as to isolate the callus. Further, a 0.5M-mannitol solution was addedto the callus to be again formed into a suspension, and the resultantproduct was moderately centrifuged, thereby to provide tobacco callusfrom which the above support (B) had been removed completely.

Example 6

With respect to each of dried Sumicagel S-50 (mfd. by Sumitomo KagakuKogyo K.K., poly (acrylic acid-vinyl alcohol) copolymer, sphericalshape, diameter=180-290 μm) and dried Aquaric CA-H (mfd. by NipponShokubai K.K., crosslinked polyacrylic acid product, indeterminate bulkshape, dimension=1-3 mm), the equilibrium water absorption was measuredin each of distilled water, physiological saline solution, and a culturemedium for a plant (MS-culture medium) at room temperature, thereby toobtain results as shown in FIG. 4 (Table 2).

Example 7

0.32 g of the dried Sumicagel S-50 used in Example 6 was added to anddispersed in 8 mL of an MS-culture medium having a sucrose concentrationof 2 wt. %, and poured into a test tube (diameter=18 mm, length=100 mm)and was left standing at room temperature. As a result, the driedSumicagel S-50 completely absorbed the culture medium to be convertedinto a gel state. The M/G ratio in this Example was 25, and was also 25%of the equilibrium culture medium absorption (100) of the Sumicagel S-50(measured in Example 6).

0.08 mL of a liquid wherein various germs had been propagated (variousgerm concentration: 1,000 germs/mL) was added onto the surface of theabove-mentioned gel. The various germ-propagated liquid was completelyabsorbed into the above gel carrier. When the culturing was conducted inthis state at about 25° C., for 3 days, the colonies of the germs wereobserved on the surface of the gel, but apparently, the penetration ofgerms into the interior of the gel was not recognized.

Example 8

0.18 g of the dried Aquaric CA-H used in Example 6 was added to anddispersed in 8 mL of an MS-culture medium having a sucrose concentrationof 2 wt. %, and poured into a test tube (diameter=18 mm, length=100 mm)and was left standing at room temperature. As a result, the Aquaric CA-Hcompletely absorbed the culture medium to be converted into a gel state.The M/G ratio in this Example was 44.4, and was also 44.4% of theequilibrium culture medium absorption (100) of the Aquaric CA-H(measured in Example 6).

0.08 mL of the various germ-propagated liquid used in Example 7 wasadded onto the surface of the above-mentioned gel. The variousgerm-propagated liquid was completely absorbed into the above gelcarrier. When the culturing was conducted in this state at about 25° C.for 3 days, colonies of the germs were somewhat observed on the surfaceof the gel, but apparently, the penetration of germs into the interiorof the gel was not recognized.

Comparative Example 3

0.064 g of agar was added to 8 mL of an MS-culture medium having asucrose concentration of 2 wt. % and then boiled so as to dissolve theagar in the culture medium. Immediately thereafter, the resultantmixture was poured into a test tube (diameter=18 mm, length=100 mm) andwas left standing at room temperature to be converted into a gel state.

0.08 mL of the various germ-propagated liquid used in Example 7 wasadded onto the surface of the above-mentioned agar gel, and cultured atabout 25° C. As a result, the liquid containing the various germs wasnot absorbed into the agar gel, and 3 days after, the propagation of thegerms covering the entire surface of the gel was observed.

Comparative Example 4

8 mL of an MS-culture medium having a sucrose concentration of 2 wt. %was poured into a test tube (diameter=18 mm, length=100 mm). 0.08 mL ofthe various germ-propagated liquid used in Example 7 was added to theculture medium, and cultured at about 25° C. As a result, 3 days after,the culturing liquid became whitely turbid, and marked propagation ofthe germs was observed.

Example 9

15 g of N-isopropyl acrylamide (NIPAAm, mfd. by Kojin K.K.), 0.47 g ofacrylic acid, 0.1 g of N,N'-methylenebis-acrylamide (Bis), 0.2 g ofammonium persulfate, 6.6 mL of 1N-NaOH, and 0.1 mL ofN,N,N',N'-tetramethylethylene diamine were dissolved in 90 mL ofdistilled water. The resultant mixture was subjected to polymerizationfor 4 hours at room temperature, thereby to obtain a poly-N-isopropylacrylamide (PNIPAAm) hydrogel having a crosslinked structure.

The resultant gel was mechanically crushed by means of a mixer, therebyto prepare indeterminately shaped blocks (C-PNIPAAm-H). The C-PNIPAAm-Hwas dispersed in one liter of distilled water and cooled to 4° C.Thereafter, the resultant mixture was warmed to 50° C. so as to shrinkthe C-PNIPAAm-H, and the resultant supernatant liquid was discarded.Such a washing operation was repeated two times, thereby to remove theunreacted monomer and the remaining polymerization initiator. Further,the C-PNIPAAm-H was dried under vacuum.

The equilibrium culture medium absorption of the thus obtainedC-PNIPAAm-H was considerably different depending on the conditionwhether the temperature was above or below the lower limit criticalsolution temperature (LCST, in the neighborhood of 30° C.) of PNIPAAm,and was about 90 at 25° C., and about 2.0 at 35° C. in a Hyponex culturemedium (Hyponex 7-6-19, mfd. by Hyponex Japan K.K.).

Example 10

In a test tube (diameter=20 mm, length=150 mm), 0.4 g of the C-PNIPAAm-Hobtained in Example 9 was mixed with and dispersed in 20 mL of a liquidmedium (having a composition as shown in FIG. 5 (Table 3)), and leftstanding at room temperature. As a result, the C-PNIPAAm-H completelyabsorbed the liquid medium to be formed into a gel state.

On the surface of the above gel, two plantlets of YT-57 (Cym. LOVELYANGEL "The Two Vergins") which had been grown so as to provide a leaflength of 4 cm were transferred, and were cultured in a culture chamber(25° C., 3000 lux, 16h-fluorescent light illumination) under anon-sterilized condition. Even after two months counted from theinitiation of the culturing, apparently, the contamination due tovarious germs was not recognized on the gel surface and in the interiorof the gel, and the plantlets were smoothly grown so as to provide aleaf length of 10 cm.

Comparative Example 5

In a test tube (diameter=20 mm, length=150 mm), 0.12 g of agar was addedto 20 mL of the liquid medium used in Example 10, and the resultantmixture was boiled so as to dissolve the agar in the culture medium, andthereafter was left standing at room temperature, thereby to form anagar gel.

On the surface of the above gel, two plantlets of YT-57 which had beengrown so as to provide a leaf length of 4 cm were transferred in thesame manner as in Example 10, and was cultured in a culture chamber (25°C., 3000 lux, 16h-fluorescent light illumination) under a non-sterilizedcondition. After one week counted from the initiation of the culturing,the propagation of various germs was recognized on the agar gel surface.After one month counted from the initiation of the culturing, thecontamination was recognized at the base portion of the plant and alongthe roots thereof which had been grown in the agar gel, and the growthof the plantlets was strongly suppressed.

Example 11

In a test tube (diameter=20 mm, length=200 mm), 0.4 g of the C-PNIPAAm-Hobtained in Example 9 was mixed with and dispersed in 20 mL of a Hyponexculture medium (Hyponex 7-6-19, mfd. by Hyponex Japan K.K, 3.5 g/L)containing 20 g/L of sucrose, and 100 g/L of banana. The resultantmixture was then sterilized by an autoclave treatment (121° C., 1.2Kg/cm², for 20 minutes), and left standing at room temperature. As aresult, the polymer completely absorbed the culture medium to be formedinto a gel state.

On the surface of the above gel, two plantlets of YT-57 which had beengrown so as to provide a leaf length of 2 cm were transferred, and werecultured in a culture chamber (25° C., 3000 lux, 16h-fluorescent lightillumination) under a sterilized condition. After three months countedfrom the initiation of the culturing, the plantlets were grown so as toprovide a length of about 10 cm. During the three-month culturing, waterwas absorbed into the plantlets or evaporated, whereby the level of theculture medium surface was lowered. When 10 mL of a Hyponex solution(Hyponex 7-6-19, 2 g/L) was further supplemented sterilizedly to thegel, the supplemented solution was completely absorbed into the abovegel, and the level of the culture medium surface was raised. Thereafter,the growth of the plantlets was markedly promoted as compared with thatin the case of a similar example provided with no supplementalfertilizer.

Comparative Example 6

In a test tube (diameter=20 mm, length=200 mm), 0.12 g of agar was mixedwith and dispersed in 20 mL of a Hyponex culture medium used in Example11, and sterilized by an autoclave treatment and the agar wassimultaneously dissolved, and left standing at room temperature, therebyto form a gel.

On the surface of the above gel, two plantlets of YT-57 (Cym, LOVERYANGEL, "The Two Vergins") which had been grown so as to provide a leaflength of 2 cm were transferred, and were cultured in a culture chamber(25° C., 3000 lux, 16h-fluorescent light illumination) under asterilized condition. After three months counted from the initiation ofthe culturing, the seedings were grown so as to provide a length ofabout 10 cm. During the three-month culturing, water was absorbed intothe plantlet or evaporated, whereby the level of the culture mediumsurface was lowered. When 10 mL of a Hyponex solution (Hyponex 7-6-19, 2g/L) was further supplemented sterilizedly to the agar gel, thesupplemented solution was not absorbed into the agar gel, and the levelof the culture medium surface was not raised. As a result, the baseportion of the plant was placed below the level of culture mediumsurface.

Example 12

In a plant box (Shibata Hario K.K., made of polycarbonate, upperpart:=75×75 mm, lower part 65×65 mm, height=100 mm), 2.1 g of theC-PNIPAAm-H prepared in Example 9 was mixed with and dispersed in 105 mLof a Hyponex culture medium used in Example 11, and was sterilized by anautoclave treatment (121° C., 1.2 Kg/cm², 20 minutes), and left standingat room temperature. As a result, the C-PNIPAAm-H completely absorbedthe culture medium to be converted into a gel state. The M/G ratiothereof was 56% of the equilibrium culture medium absorption of theC-PNIPAAm-H.

On the surface of the above gel, 16 plantlets of YT-57 which had beengrown so as to provide a leaf length of 2 cm were transferred, and weresterilizedly cultured in a culture chamber (25° C., 3000 lux,16h-fluorescent light illumination). After three months counted from theinitiation of the culturing, when the plantlets were grown so as toprovide a length thereof of about 10 cm, the plant box wasnon-sterilizedly immersed in warm water at 35° C. for 20 minutes. As aresult, the C-PNIPAAm-H was shrunk and almost all of the culture mediumwhich had been contained in the carrier was discharged from the carrier.

The lid of the plant box was opened and the above discharged culturemedium was sucked by using a dropping pipette, and then about 150 mL ofaqueduct water at a temperature of about 16° C. was added to the plantbox, whereby the aqueduct water was absorbed into the C-PNIPAAm-H. Thetemperature was again raised to about 35° C. to shrink the C-PNIPAAm-H,whereby the aqueduct water was discharged from the carrier. When thesugar concentration of the discharged aqueduct water was measured bymeans of a refractometer, the content was found to be below thedetection limit thereof (0.2 wt. %).

The discharged aqueduct water was removed by using a dropping pipette,and about 105 mL of a liquid medium used in Example 10 was added to theplant box. Then, the plant box was non-sterilizedly subjected toculturing in a culture chamber (25° C., 3000 lux, 16h-fluorescent lightillumination) under a condition such that the lid of the plant box wasleft open and the plant box was covered with a vinyl bag in which a holehaving a diameter of 5 mm had been opened. Even after one month countedfrom the initiation of the culturing, the contamination of various germswas not recognized apparently, and the plantlets were smoothly grown.

At this time, the vinyl bag was taken off, and the plant box wasimmersed in warm water of 35° C. for 20 min. in the same manner asdescribed hereinabove. As a result, the C-PNIPAAm-H was shrunk anddischarged almost all of the culture medium which had been contained inthe above carrier.

The discharged culture medium was removed, and about 105 mL of theliquid medium used in Example 10 was added to the plant box, and theplantlets were grown in a greenhouse, while aqueduct water wassupplemented at intervals of 2-3 days with respect to the vaporizedportion of water from the upper portion of the vessel. The plantletswere smoothly grown even after about 1 month, but the algal generationon the gel surface was recognized by the naked eye when the period ofthe culturing exceeded about 1.5 month. However, the plantlets showedsmooth growth substantially without receiving the influence of the alga.

Further, at a point of time at which the period of the growth in thevinyl greenhouse exceeded 2 months, the plantlets were taken out fromthe above plant box, and transferred to a black vinyl pot having adiameter of about 9 cm, while Growell MO-2 (bark produced in NewZealand, available from Mukoyama Orchid Ltd.) was disposed around theplantlets, in a state such that the culture medium was as such attachedto the roots of the plantlets. Thereafter, the pot was subjected toordinary cultivation in a greenhouse.

After 3 months, when the state of the growth of the upper portion of theplant above the ground was investigated, it was found that the leafwidth was large, the color of the leafs was thick, and further the baseportion was thick, and the growth of the plantlets was very good.

Comparative Example 7

In a plant box used in Example 12, 0.63 g of agar and 105 mL of aHyponex culture medium used in Example 12 were charged, and sterilizedby an autoclave treatment, and left standing at room temperature,thereby to be formed into a gel.

On the surface of the above gel, 16 plantlets of YT-57 which had beengrown so as to provide a leaf length of 2 cm were transferred, and werecultured in a culture chamber (25° C., 3000 lux, 16h-fluorescent lightillumination) under a sterilized condition. After three months countedfrom the initiation of the culturing, the plantlets were grown so as toprovide a length of about 10 cm. At this time, the lid of the plant boxwas opened and the culturing was conducted non-sterilizedly. As aresult, after three days counted from the opening of the lid, thecontent in the plant box was contaminated with various germs. After oneweek, remarkable propagation of various germs was observed in most partof the agar gel and the portion around the base portion of the plant.

Example 13

In a Pack-Plast container (Tomohiro Trade K.K., cylindrical plasticvessel, inside diameter of upper part=110 mm, inside diameter of lowerpart=90 mm, height=75 mm), 4 g of the dried C-PNIPAAm-H prepared inExample 9 was mixed with and dispersed in 200 mL of a Hyponex solution(Hyponex 20-20-20, Hyponex Japan K.K., 1 g/L), whereby the C-PNIPAAm-Hcompletely absorbed the solution to be formed into a gel state.

In the resultant gel, ten plantlets of an orchid, "RG310" (Cym. ENZANSYMPHONY "RG310") which had been grown under a sterilized condition soas to provide a fresh weight of 0.25 g were transferred, were grown in agreenhouse under a condition such that the upper part of the Pack-Plastcontainer was completely opened (i.e., under a non-sterilizedcondition). During this growth, a solution of the above Hyponex whichhad been diluted so as to provide double volume, as an additionalfertilizer was supplied to the container from the upper part of thecontainer so as to supplement a nutrient or water to be vaporized fromthe upper part of the container or to be absorbed into the plant.

The growth of the portion of the plantlet below the gel surface wassomewhat slow, but the portion thereof above the gel surface wassmoothly grown. FIG. 6 (Table 4) shows the results obtained by measuringthe degree of the growth (fresh weight) of the plantlet after 48 dayscounted from the initiation of the cultivation.

Example 14

In a Pack-Plast container, 2 g of the dried C-PNIPAAm-H prepared inExample 9 was mixed with and dispersed in 100 mL of a Hyponex solutionused in Example 13 in the same manner as in Example 13, whereby theC-PNIPAAm-H completely absorbed the solution to be formed into a gelstate. Then, 100 mL of Growell MO-2 was impregnated with a large excessof a Hyponex solution, and then a part of the solution (gravitationalwater) which flowed out due to gravity was removed, and the resultantGrowell was mixed into the above gel.

In the same manner as in Example 13, ten plantlets of an orchid, "RG310"which had been grown so as to provide a fresh weight of 0.25 g weretransferred onto the resultant support (mixture of C-PNIPAAm-H andGrowell MO-2) prepared above, and the plantlets were grown in agreenhouse under a condition such that the upper part of the Pack-Plastcontainer was completely opened (i.e., under a non-sterilizedcondition). During this growth, a solution of the above Hyponex whichhad been diluted so as to provide double volume, as an additionalfertilizer was supplied to the container from the upper part of thecontainer so as to supplement a nutrient or water to be vaporized fromthe upper part of the container or to be absorbed into the plant.

Both of the portions of the above plantlets (portions thereof below thegel surface and above the gel surface) were smoothly grown. When thefresh weights of the plantlets after 48 days counted from the initiationof the cultivation were measured, the results as shown in FIG. 6 (Table4) were obtained.

Comparative Example 8

200 mL of Growell MO-2 was charged into a Pack-Plast container used inExample 13, and was impregnated with a large excess of a Hyponexsolution, and then a part of the solution (gravitational water) flowingout due to gravity was removed.

In the same manner as in Example 13, ten plantlets of an orchid, "RG310"which had been grown so as to provide a fresh weight of 0.25 g weretransferred onto the resultant support, and the plantlets were grown ina greenhouse under a condition such that the upper part of thePack-Plast container was completely opened, i.e., under a non-sterilizedcondition. During this growth, a solution of the above Hyponex which hadbeen diluted so as to provide double volume, as an additional fertilizerwas supplied to the container from the upper part of the container so asto supplement a nutrient or water to be vaporized from the upper part ofthe container or to be absorbed into the plant.

The above plantlets were slowly grown. When the fresh weights of theplantlets after 48 days counted from the initiation of the cultivationwere measured, the results as shown in FIG. 6 (Table 4) were obtained.Industrial Applicability

As described hereinabove, according to the present invention, there isprovided a support for growing or regenerating a plant, comprisingparticles having a dimension in the range of 0.1 μm to 1 cm in a driedstate, and comprising a hydrogel having a crosslinked structure.Particles of the crosslinked hydrogel having of the above-mentionedspecific dimension have a property such that they are swollen in wateror a culture medium so as to reversibly increase their volume to beconverted into a gel state, and therefore various plants may besupported in the resultant gel and the plants therein may be grown orregenerated. On the other hand, when the plant is intended to becollected, an excess of water or culture medium is added to the gel soas to decrease the ratio of the volume of the support to that of theculture medium, whereby the plant-supporting ability of the support maybe decreased and the above plant may easily be separated from thesupport.

When the above support is used, the embedding of the plant into the gel,the recovery of the plant from the gel, etc., may easily be conductedwithout damaging the plant at all, by regulating the volume ratio of thesupport to the water or culture medium. Therefore, according to thepresent invention, it is possible to solve the problems of theconventional gel culturing process using agar, etc.

Further, the present invention also provides a support for growing orregenerating a plant, comprising a polymer which has been obtained bycrosslinking a temperature-responsive polymer having a LCST (lowercritical solution temperature).

When the above support is used, the embedding of the plant into the gel,the recovery of the plant from the gel, etc., may easily be conductedwithout damaging the plant at all, e.g., by changing the temperature ina physiological temperature range of the plant. Therefore, according tothe present invention, it is possible to completely solve the problemsof the conventional gel culturing process using agar, etc.

The present invention further provides a carrier for growing orregenerating a plant, comprising a culture medium, and a polymer,wherein the culture medium is absorbed in and retained by the networkstructure comprising the above polymer substantially completely.

When the above carrier according to the present invention is used, it ispossible to grow or regenerate a plant while effectively suppressing thepropagation of bacteria and fungi, whereby the problems of theconventional gel culturing process using agar may be solved.

I claim:
 1. A carrier for growing or regenerating a plant, comprising apolymer which has been obtained by crosslinking a temperature-responsivepolymer having a LCST (lower critical solution temperature).
 2. Thecarrier for growing or regenerating a plant according to claim 1,wherein the LCST is higher than 0° C. and not higher than 60° C.
 3. Thecarrier for growing or regenerating a plant according to claim 1, whichis in the form of: particles, micro-beads, fibers, flakes, a sponge, afilm or a plate.
 4. The carrier for growing or regenerating a plantaccording to claim 1, which has a dimension in the range of 0.1 μm to 1cm.
 5. A method of growing or regenerating a plant,comprising:dispersing a carrier for growing or regenerating a plant,which has been obtained by crosslinking a temperature-responsive polymerhaving a LCST (lower critical solution temperature), in a predeterminedculture medium at a temperature higher than the LCST; mixing a plant inthe resultant dispersion; and lowering the temperature to a value lowerthan the LCST to reduce the fluidity of the dispersion and to convertthe dispersion into a gel state, thereby to grow or regenerate theplant.
 6. A method of growing or regenerating a plant,comprising:dispersing a carrier for growing or regenerating a plant,which has been obtained by crosslinking a temperature-responsive polymerhaving a LCST (lower critical solution temperature), in a predeterminedculture medium at a temperature higher than the LCST; lowering thetemperature to a value lower than the LCST to reduce the fluidity of theresultant dispersion and to convert the dispersion into a gel state; anddisposing or inserting a plant on or in the gel, thereby to grow orregenerate the plant.
 7. The method of growing or regenerating a plantaccording to claim 5, wherein the gel containing the grown orregenerated plant is again converted into a dispersion liquid of thecarrier at a temperature higher than the LCST, thereby to recover and/ortransfer the plant.
 8. The method of growing or regenerating a plantaccording claim 5, wherein the plant which has been grown or regeneratedat a temperature lower than the LCST is recovered and/or transferredfrom the dispersion liquid at a temperature higher than the LCST; thecarrier is separated and recovered from the dispersion liquid; andthereafter the carrier is washed and sterilized, thereby to reuse therecovered carrier as a carrier for growing or regenerating a plant.
 9. Asupport for growing or regenerating a plant, comprising a networkstructure material and a culture medium retained in the networkstructure material in a proportion of 10% to 100% of the equilibriumculture medium absorption; said network structure material having adimension in the range of 0.1 μm to 1 cm in a dried state, andcomprising a hydrogel having a crosslinked structure.
 10. The supportfor growing or regenerating a plant according to claim 9, wherein agrown or regenerated tissue of the plant does not penetrate into theinterior of the hydrogel having a crosslinked structure.
 11. The supportfor growing or regenerating a plant according to claim 9, wherein thenetwork structure material is in the form of: particles, micro-beads,fibers, flakes, a sponge, a film or a plate.
 12. A method of growing orregenerating a plant, wherein a support is swollen with a culture mediumin a culturing vessel so as to reduce the fluidity of the culture mediumto be formed into a gel state, and to support a plant by the gel,thereby to grow or regenerate the plant, wherein the plant is grown orregenerated by using the support, and thereafter an excess of water isadded to the support to increase the fluidity of the support, thereby torecover the plant, and the support comprises particles having adimension in the range of 0.1 μm to 1 cm in a dried state, andcomprising a hydrogel having a crosslinked structure.
 13. A carrier forgrowing or regenerating a plant, comprising a culture medium and apolymer, wherein the culture medium is substantially absorbed into andretained by the network structure comprising the polymer.
 14. Thecarrier for growing or regenerating a plant according to claim 13,wherein the network structure comprises a hydrogel having a crosslinkednetwork structure which prevents the penetration thereinto of bacteria,fungi and/or a regenerated tissue of plant.
 15. The carrier for growingor regenerating a plant according to claim 13, wherein the networkstructure material has a dimension in the range of 0.1 μm to 1 cm in adried state, and the carrier is in the form of particles, micro-beads,fibers, a film, or a sheet-like shape.
 16. A method of growing orregenerating a plant, wherein a carrier comprising a culture medium anda polymer constituting a network structure is used so as to grow orregenerate a plant while suppressing the propagation of bacteria andfungi; the culture medium being substantially absorbed into and retainedby the network structure in a proportion of 10% to 100% of theequilibrium culture medium absorption of the polymer constituting thenetwork structure.
 17. The method of growing or regenerating a plantaccording to claim 6, wherein the gel containing the grown orregenerated plant is again converted into a dispersion liquid of thecarrier at a temperature higher than the LCST, thereby to recover and/ortransfer the plant.
 18. The method of growing or regenerating a plantaccording to claim 6, wherein the plant which has been grown orregenerated at a temperature lower than the LCST is recovered and/ortransferred from the dispersion liquid at a temperature higher than theLCST; the carrier is separated and recovered from the dispersion liquid;and thereafter the carrier is washed and sterilized, thereby to reusethe recovered carrier as a carrier for growing or regenerating a plant.